Method for preparing entomopathogenic nematodes for storage by mixing with non-fibrous cellulose particles of less than 300 μM

ABSTRACT

Third stage juvenile (J3) entomopathogenic nematodes are prepared for storage by being induced into a state of cryptobiosis. The induction of cryptobiosis is effected by mixing an aqueous cream of the J3 nematodes with anhydrous, small particles (average maximum dimension less than 300 μm) of non-fibrous cellulose. The proportions of the aqueous cream and non-fibrous cellulose particles are such that, after equilibration, the mixture has a water activity in the range 0.80 to 0.995. Preferably an anti-fungal agent is included in the aqueous cream. To store the cryptobiotic J3 nematodes, the mixture is preferably kept in a container, fitted with an attachment which maintains the water activity in the container at a required value. The attachment includes a rigid tube that connects the interior of the container with a chamber that is vented to ambient atmosphere by small apparatus. When in use the chamber contains water-absorbent material saturated with water or with a saturated salt solution, and the tube contains an air-permeable plug. An alternative attachment comprises a plastic envelope, one face of which is stuck to the wall of the container. Small apertures in the face are aligned with apertures in the container wall. Small apertures in the other face connect the inside of the envelope to ambient atmosphere. In use, the envelope contains a water-absorbent material saturated with water or a salt solution, and at least one spacer member.

TECHNICAL FIELD

The invention concerns the storage of entomopathogenic nematodes fortransport or future use. More particularly it concerns the preparationfor storage, and also the storage, of the third stage infectivejuveniles (commonly called “J3” nematodes) of nematodes belonging to thegenera Steinernema (synonym Neoaplectana) and Heterorhabditis (synonymChromonema), at controlled water activities.

BACKGROUND

It is well known that entomopathogenic nematodes in the familiesSteinernematidae and Heterorhabditidae have considerable potential forthe biological control of a number of insect pests. Infective thirdstage juveniles (J3) of the nematodes (which can survive many weeks inthe environment without feeding) are able to seek out an insect,penetrate into the insect's haemocoel and there release specificsymbiotic bacteria (Xenorhabdus or Photorhabdus species). The bacteriakill the insect within a day or so and provide suitable conditions forthe juvenile nematodes to progress to the adult stage, and for nematodereproduction.

The specification of International Patent Application No.PCT/AU93/00465, which was published as WIPO Publication No. WO 94/05150,contains a comprehensive summary of previously adopted methods for, anddetails of what was then recent work in, the storage of J3entomopathogenic nematodes, including a description of an effectivetechnique for the preparation for relatively long term storage of J3nematodes. That technique involved mixing together an aqueousconcentrate of clean J3 entomopathogenic nematodes and substantiallyanhydrous particles of a highly water-absorbent material, theproportions of the aqueous concentrate and water-absorbent material inthe mixture being such that (a) sufficient water is absorbed from theconcentrate by the absorbent particles to induce cryptobiosis of thenematodes, and (b) the mixture, after equilibrating, has a wateractivity in the range of from 0.80 to 0.995. Suitable arrangements forstoring the cryptobiotic J3 nematodes at the selected water activityvalue are also described in WIPO Publication No. WO 94/05150.

DISCLOSURE OF THE PRESENT INVENTION

The first aspect of the present invention is an improvement in what hasbeen, up to now, the preferred technique for preparing J3entomopathogenic nematodes for storage, as described in WIPO PublicationNo. WO 94/05150, namely the selection of the water-absorbent material tobe used in that method to induce cryptobiosis of the J3 nematodes, andto carry the J3 entomopathogenic nematodes that are in a state ofinduced cryptobiosis.

In WIPO Publication No. WO 94/05150, it is shown that polyacrylamide gelparticles, or particles of starch polyacrylamide gel (optionally withpolyacrylamide gel particles also present), or methyl cellulose powder,may be used as the medium both for use in the induction of the state ofnematode cryptobiosis and for maintaining a selected water activityduring subsequent storage.

In fact, methyl cellulose is not a highly water-absorbent compound andits recitation in WIPO Publication No. WO 94/05150 was inappropriate(being based on anecdotal evidence of methyl cellulose as a waterabsorbent). Methyl cellulose, which is used as an appetite suppressor,is a chemically derived compound which is soluble in water and which, inthe course of dissolving, expands to form a glue-like product. Althoughanhydrous particles of methyl cellulose can absorb water from an aqueouscream of J3 entomopathogenic nematodes to induce cryptobiosis of thenematodes and establish a mixture that has the desired water activity,(a) the glue-like mass in which the J3 nematodes are distributedprevents air (oxygen) from reaching the nematodes, and (b) if,subsequently, water is added to the mixture of J3 nematodes, water andmethyl cellulose, to release the nematodes for spraying, the methylcellulose, being in solution, cannot be removed by sieving and theglue-like consistency of the mixture clogs the spray. nozzles. In fact,it is now clear that it is impractical to use anhydrous particles ofmethyl cellulose in the method described and claimed in WIPO PublicationNo. WO 94/05150.

Anhydrous particles of polyacrylamide gel, and of starch polyacrylamidegel, are significantly better as a storage substrate than attapulgiteclay, which has been used previously for this purpose. This is primarilybecause, when particles of polyacrylamide gel, and of starchpolyacrylamide gel, are used as described in WIPO Publication No. WO94/05150, air is available to the nematodes while in their cryptobioticstate and it is not necessary to cool the nematodes during theirstorage. When attapulgite clay is used as the storage medium, a layeredand compressed mixture of clay and cryptobiotic J3 nematodes is formed.This compressed mixture restricts the availability of air to thenematodes. This mixture, therefore, has to be kept in a refrigeratorduring storage to reduce the activity (and hence reduce the oxygenintake requirements) of the nematodes. However, the particles ofpolyacrylamide gel, or of starch polyacrylamide gel, retain a lot ofwater, particularly when a water activity of about 0.995 is establishedfor the mixture. At this water activity level, particles ofpolyacrylamide gel, or of starch polyacrylamide gel, retain about 300times their weight of water, which is really waste material.

There is one (quite old) recorded use of cellulose, in the form of afilter paper, as a medium on which J3 entomopathogenic nematodes can bestored. That disclosure is in the paper by J. F. Howell, entitled “Newstorage methods and improved trapping techniques for the parasiticnematode Neoaplectana carpocapsae”, which was published in the Journalof Invertebrate Pathology, Volume 33, pages 155 to 158, 1979. Howell puthis J3 nematodes on a filter paper into a cold environment, thusensuring that they did enter into a protected state, similar tocryptobiosis. However, there was no control of water activity and the J3nematodes would not have survived if stored at a higher temperature.Moreover, storage of nematodes on a filter paper would not be acommercial proposition. If the nematodes should survive the storageperiod, they would be difficult to disperse in the field. The fibrouscellulose in a filter paper clogs up a spray nozzle, even when thefilter paper is cut into small pieces. Presumably, to disperse thenematodes, they would have to be formed into a sprayable aqueoussuspension by (i) immersing the filter papers containing the storednematodes in water until the nematodes become active and leave thefilter paper, then (ii) removing the filter papers from the dilutesuspension of nematodes thus obtained, and (iii) optionallyconcentrating the suspension by decantation after allowing the nematodesto settle, or by collection of the nematodes using a muslin cloth, toform a sprayable suspension of J3 nematodes.

The present inventors have discovered that small particles (that is,particles having a size of less than 300 microns (300 μm), preferablyless than 200 microns, more preferably less than about 100 microns, andmost preferably less than 50 microns) of non-fibrous cellulose can beused as a storage medium for J3 entomopathogenic nematodes and that amixture of J3 nematodes and such particulate cellulose, when suspendedin water, is sprayable. The small particles of cellulose, unlikeanhydrous particles of methyl cellulose, are not soluble. Celluloseparticles are not highly water-absorbent (they absorb about 2.2 timestheir weight of water when establishing a water activity of about0.995). When small anhydrous particles of cellulose are mixed with anaqueous cream of J3 entomopathogenic nematodes to induce cryptobiosis ofthe nematodes and establish a desired water activity, a fluffy mixtureis produced, in which air (oxygen) is available to the J3 nematodes inthe mixture. Thus the mixture (a) does not include a large amount ofwaste water (which is the case when particles of polyacrylamide gel, orof starch polyacrylamide gel, are used), and (b) does not have to becooled during storage to reduce the activity of the nematodes (which isnecessary when the cryptobiotic nematodes are stored in a layeredmixture with attapulgite clay). Furthermore, it has been found that J3nematodes stored in this manner (preferably with the addition of anantifungal agent) can be distributed in the field simply by adding thestored mixture of nematodes (in a state of cryptobiosis) to clean water,waiting for a period of from 5 minutes to one hour for the nematodes toreach their full activity level, then spraying the suspension ofnematodes and particulate cellulose.

Experiments conducted by the present inventors have shown that J3entomopathogenic nematodes can be stored effectively for extendedperiods by using the method described in WIPO Publication No. WO94/05150, with small diameter particulate non-fibrous cellulose materialsubstituted for the highly water-absorbent material of that method.

Hence, according to the first aspect of the present invention, a methodof preparing third stage infective juveniles (J3) of entomopathogenicnematodes for storage comprises: mixing together an aqueous concentrateof clean J3 entomopathogenic nematodes and substantially anhydrous smallparticles of non-fibrous cellulose, the particles having an averagemaximum dimension which is less than 300 μm, the proportions of theaqueous concentrate and particulate cellulose in the mixture being suchthat (a) sufficient water is absorbed from the concentrate by thecellulose particles to induce cryptobiosis of the nematodes, and (b) themixture, after equilibrating, has a water activity in the range of from0.80 to 0.995.

As noted above, the preferred maximum dimension of the celluloseparticles is less than 200 microns and more preferably less than 100microns. Most preferably the cellulose particles have a diameter of lessthan 50 microns. A particularly useful form of non-fibrous cellulose isthe BO-2 grade cellulose available from Cellulose-Füllstoff-Fabrik GmbH& Co KG, generally known as “CCF”, of Mönchengladbach, Germany, underthe trade mark TECHNOSEL 40. That BO-2 grade cellulose, it is believed,is produced by milling a cellulose pulp that has been treated to removelignins and other impurities. It consists of cellulose particles whichhave a generally rectangular cross-section with an average maximumdimension of about 32 microns (32 μm).

Preferably the water activity of the equilibrated mixture of J3nematodes, particulate cellulose and water is in the range of from 0.92to 0.995, and more preferably the water activity of the equilibratedmixture is from 0.95 to 0.99.

Preferably, an anti-fungal agent is included in the mixture of J3nematodes, non-fibrous cellulose particles and water.

Embodiments of the first aspect of the present invention will now bedescribed, by way of example only.

DESCRIPTION OF EMBODIMENTS OF THE PRESENT INVENTION

In a series of trials conducted by the present inventors to confirm theefficiency of the present invention, samples of third stage juvenileentomopathogenic nematodes were reared and extracted using the methodsdescribed by R A Bedding, M S Stanfield and G W Crompton in thespecification of International patent application No. PCT/AU91/00136(WIPO Publication No. WO 91/15569). However, to perform this inventionthe nematodes may be reared on insects in vivo or in liquid culture,provided the nematodes (a) are free from appreciable amounts ofextraneous matter remaining from the culture medium, and (b) arerelatively free from nematode stages other than J3 (preferably no adultnematodes are present and certainly no more than 2 per cent of thenematodes should be adults).

The entomopathogenic nematodes reared for the trials by the presentinventors, as in the technique described in the specification ofInternational patent application No. PCT/AU88/00127, were sedimentedafter washing and the excess water was drained off. The sediment ofnematodes was then pumped from settling tanks into sieves lined withcloth through which the water, but not the nematodes, could pass. Waterwas drained off in this way and further water was removed by stirringthe nematode cream while draining. In some trials, still further wasremoved by gathering up the cloth edges to enclose the nematode mass inthe cloth before squeezing out some of the remaining water. Theresulting cream of nematodes contained from 0.5 to 3.5 million J3nematodes per gram, depending upon the species involved and the amountof inter-nematode water remaining. For some experiments, one of variousantifungal agents was added to and mixed with the sedimented nematodeswhile they were still in the tanks. Hence, after removal of much of thesurface water (and thus also of most of the antibiotic and/or antifungalagent), some of the antifungal agent remained to be absorbed by thecellulose particles in the induction of cryptobiosis process. Theantifungal agents used by the present inventors were copper oxychlorideand those marketed under the trade marks Amistar (a brand ofazoxystrobin), Benlate, Tecta and Proxel (Some of these fungicides alsohave bactericidal properties.)

It will be appreciated that methods of rearing the J3 nematodes otherthan those mentioned above may be used, and that this list of fungicidesis not exhaustive but represents the anti-fungal agents used by thepresent inventors.

The technique usually adopted for combining the nematodes and theabsorbent material was as follows. The cellulose particles were weighedand added to the appropriate weight of nematodes. (The appropriateweights were determined by prior experimentation to ascertain whichcombinations fall within the required range of water activities). Thecellulose particles and nematodes were then immediately stirred andmixed together so that the cellulose particles were evenly distributedin the mixture.

The water activity of the mixture of nematodes and cellulose particleshas to be in the range of from 0.80 to 0.995. As noted above, the wateractivity is preferable in the range of from 0.92 to 0.995, and mostpreferably is in the range of from 0.95 to 0.99.

The required water activity of the mixture is attained quickly, but notimmediately. The cellulose particles take up the free surface water ofthe nematode cream instantaneously, and then absorb water that isreleased from within the nematodes. Thus, when the free surface water isfirst taken up, the mixture of J3 nematodes, cellulose particles andwater has a water activity lower than its final value, which is attainedwithin one or two hours.

The normal procedure adopted by the present inventors after mixingtogether a nematode cream and a quantity of anhydrous celluloseparticles is to leave the mixture overnight at a temperature in therange of from 15° C. to 23° C. in conditions allowing for aeration butwith reduced evaporation. This was. usually achieved by keeping themixture in a covered container. After this overnight storage period,samples from the mixture of cellulose particles (now swollen with water)and nematodes are placed in a variety of storage containers, each withprovision for gaseous exchange between the interior and the exterior ofthe container (while minimising water loss), so that anaerobicconditions cannot develop within the container.

Now the value of the present invention is in the extended storage of J3nematodes that it enables. To test this storage period, samples of theequilibrated mixture containing J3 nematodes in a stage of cryptobiosiswere placed in various containers. Some of these containers had positiveventilation arrangements (for example, a series of holes in thecontainer). Others were containers which were provided with a membrane,or included a panel, of a material through which air can permeate.

However, new forms of storage container were developed in conjunctionwith the present invention and those (preferred) new containersconstitute the second aspect of the present invention, which isdescribed below.

In some experiments, the containers were stored at the extended storagetemperature of the experiment immediately after receiving a sample ofthe equilibrated mixture of nematodes, water and non-fibrous celluloseparticles. In other experiments, the containers were stored firstly at15° C. for three days and then at the extended storage temperature. Inall experiments, the extended storage was effected in a manner such thatthe water activity of the cellulose/nematodes combination was maintainedat a value in the range of from 0.80 to 0.99.

It was noted early in these trials that if the stored J3 ii nematodeswere affected by a fungus, the storage period was reduced significantly.To prevent a fungus infection, a fungicide was included in many of thesamples tested. Of the fungicides mentioned above, azoxystrobin waspreferred, preferably in combination with Proxel (trade mark).

A particularly severe test of the present invention was storage of thecryptobiotic nematodes at 23° C. Normally, it is difficult to store J3nematodes for more than 2 to 3 weeks at this temperature. Using thepresent invention, the following storage times were achieved for J3nematodes:

Steinernema glaseri—From 2 to 5 months

Steinernema feltiae—From 4 to 5 months

Steinernema carpocapsae—About 8 months

Steinernema scapterisci—About 9 months

All Heterorhabditis species tested—From 2 to 3 months.

As noted above, cryptobiotic J3 nematodes that have been stored usingthe method of the present invention can be prepared for distribution inthe field simply be adding the stored mixture of nematodes andnon-fibrous cellulose to water. The time taken for the nematodes toreach their full activity level depends upon the period for which theyhave been stored in a cryptobiotic state. Nematodes that have beenstored for a long period of time may take up to one hour to regain theirfull activity level. However, most species of nematodes regain theirfull activity level within 5 to 10 minutes. The active J3 nematodes,with their storage medium, may then be sprayed in the usual manner, forsmall particles of non-fibrous cellulose do not clog a spray nozzle.

DISCLOSURE OF THE SECOND ASPECT OF THE INVENTION

The second aspect of the present invention concerns the equipment inwhich J3 entomopathogenic nematodes in a state of cryptobiosis may bestored.

For long term storage of cryptobiotic entomopathogenic J3 nematodes, thewater activity of the storage environment must be maintained atsubstantially the required value, and there must be oxygen (air) presentfor the nematodes to respire properly. Allowing adequate aerationresults in loss of water from the storage environment. With long termstorage, the reduction of water reduces the water activity of thestorage environment. This, in turn, results in further desiccation ofthe J3 nematodes with consequent adverse effects on the nematodes, andtheir subsequent mortality.

Clearly, it will be advantageous to conserve water in the storageenvironment while maintaining the appropriate value of water activity,and this is the objective of the second aspect of the present invention.

To achieve this objective, the second aspect of the present inventionprovides a water activity control attachment for fitting to an apertureof a container to be used for storage of J3 entomopathogenic nematodesin a state of cryptobiosis.

In one form, this attachment comprises a generally rigid tube which:

(a) is adapted to be fitted in an air-tight manner in or over anaperture of the container so that one end of the tube is within thecontainer; and

(b) has its other end within a chamber which has at least one smallaperture therein which connects the inside of the chamber to the outsideair.

When in use to provide water activity control, the tube will contain aplug of an air-permeable material, such as cotton wool, and the chamberwill contain a water-absorbent material that has been saturated withwater, or with a saturated salt solution if a specific water activitywithin the storage container is required. For example, if a wateractivity of 0.97 is to be established, the water-absorbent material maycontain a saturated solution of potassium sulphate.

A modified form of this new water activity control attachment has beendesigned to fit within, and be supported by, the neck of a wide-neckedjar which is to be used to store J3 nematodes which are in a state ofcryptobiosis. This modified form of water activity control attachmentcomprises:

(a) an annular chamber adapted to fit within the neck region of awide-necked jar, the outer diameter of the annular chamber beingslightly less than the inner diameter of the neck of the jar; saidchamber being open at its top and closed at its base, whereby thecentral portion of the annular chamber forms a tube coaxial with theneck of said chamber;

(b) a flange extending horizontally outwardly from the top periphery ofsaid chamber, said flange having dimensions such that said flange isadapted to be supported by the upper surface of said neck when saidchamber is placed within said neck; and

(c) a cap adapted fit over said neck, said cap having at least one smallaperture therein.

When this form of attachment is in use, a plug of air-permeable materialis inserted into the tube at the centre of the chamber and awater-absorbent material which has been saturated with water or with aconcentrated salt solution is placed within the annular chamber.

Another form of water activity control attachment for a container to beused to store J3 entomopathogenic nematodes in a state of cryptobiosishas been devised. This third form of the second aspect of the presentinvention provides a water activity control attachment for use with acontainer having at least one aperture in the (or a) wall thereof, saidattachment comprising a plastic envelope having a front face and a rearface, a layer of adhesive being applied over at least the region of therear face which is adjacent to the edge of the rear face; the rear facehaving at least one aperture therein; and the front face of saidenvelope having at least one small aperture in the upper region thereof.

When this attachment is used to control the water activity ofcryptobiotic J3 entomopathogenic nematodes stored within the container,the envelope is attached, using the adhesive layer, to the wall of thecontainer, that wall having at least one aperture in it, with theaperture (or apertures) in the rear face of the envelope at leastpartially overlapping the aperture (or apertures) in the container wall.In addition, the envelope will contain a water-absorbent material whichhas been saturated with water or with a saturated salt solution, and mayalso contain at least one flexible spacing member to ensure that thefront and rear faces of the envelope are separated from each other andthe aperture(s) in the front face of the envelope does (do) not overlapthe aperture(s) in the rear face of the envelope.

Embodiments of these forms of water activity control attachment will nowbe described, by way of example only, with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is partly schematic sectional view of a container being used tostore cryptobiotic J3 nematodes, fitted with an embodiment of the firstform of water activity control attachment.

FIG. 2 illustrates, also in the form of a partly schematic sectionalview, a container being used to store J3 entomopathogenic nematodes, thecontainer including a modified form of the water activity controlattachment illustrated in FIG. 1.

FIG. 3 is a schematic sectional view of a container within whichcryptobiotic J3 nematodes and their support medium are to be stored,incorporating an embodiment of the third form of novel water activitycontrol attachment.

In the embodiment illustrated in FIG. 1 of the accompanying drawing, acontainer 10, having a large aperture 11 in its upper surface, is closedby a screw cap 12. The tube 13 of the water activity control attachmentof the present invention is sealed into an aperture which has been cutinto the screw cap 12. If the aperture 11 of the container 10 isnormally closed by a rubber bung, the tube 13 would be an air-tight fitin an aperture cut into that bung.

The lower end of the tube 13 is within the container 10. The upper endof the tube 13 extends into—and is sealed into—a cylindrical chamber 14,the upper wall of which comprises a screw cap 15. The screw cap 15contains a number of ventilation apertures 16. If the screw cap 15 isreplaced by a suitable lid (which will always be the case if the chamber14 is not cylindrical), that lid will be provided with the air holes(ventilation apertures) 16.

The chamber 14 contains water-saturated polyacrylamide gel particles 18(or particles of another suitable water-absorbent material) around thelower region of that part of the tube 13 that is within the chamber 14,to maintain a water activity of 1.00 in the air within the chamber 14.If a saturated salt solution is used to saturate the polyacrylamide gelparticles, a different required water activity will be established andmaintained within the chamber 14 and—by transfer of air along the tube13—within the container 10.

Although use of a saturated salt solution enables the establishment of aselected water activity within the chamber 14 and the container 10, thepresent inventors have discovered that no change in the maximum storageperiod of any species of entomopathogenic nematode is detectable if thewater activity within the container 10 is maintained at 1.00 instead ofat the ideal value of, say, 0.96. It is believed that this observationshows that the water activity within a sample 29 of J3 entomopathogenicnematodes stored in a cryptobiotic state with water and awater-absorbent medium (preferably non-fibrous cellulose particles)varies very little unless there is a significant difference between thewater activity within the sample and the water activity of thesurrounding air.

The tube 13 contains a plug 17 of cotton wool, or other suitablematerial which permits transfer of air into and out of the container 10,but which prevents microbes and small bugs, dust particles and otherparticulate contaminant material from entering the container 10.

Variations of the arrangement illustrated in FIG. 1 of the accompanyingdrawing can be made to suit individual storage containers. For example,if the storage container should have a side aperture instead of a topaperture, the lower part of the tube 13 (that is, that part of the tube13 that is below the base of the chamber 14) would be longer and wouldbe bent so that the tube could enter the container 10 substantiallyhorizontally, with the chamber 14 performing its normal function as anon-spill water reservoir.

A modified form of the water activity control equipment is illustratedin FIG. 2. In this drawing, a sample 29 of J3 entomopathogenic nematodesis stored within a container 20. The container 20 has a wide neck 21which is closed by a lid or cap 15. The cap 15 may be screwed onto theneck 21 or it may be a snap-fit over the neck 21. As in the embodimentillustrated in FIG. 1, the cap 15 in FIG. 2 is provided with a number ofsmall ventilation apertures 16. An annular chamber 24 is supportedwithin the neck region of the container 20.

The annular chamber 24 has a substantially vertical outer side wall 25and a substantially vertical inner side wall 26. The. outer diameter ofthe side wall 25 is slightly less than the inner diameter of the neck21. The walls 25 and 26 are joined by a circular web portion 27. Thechamber 24 is open at its top and the inner side wall 26 forms a tube 23which contains an air-permeable plug 17. A flange 22 extends outwardly,horizontally, from the top of the side wall 25 for a distance such that(a) the flange 22 overlaps the top surface of the neck 21, and (b) theouter diameter of the flange 22 is less than the inside diameter of theside wall of the cap 15. The annular chamber 24 contains awater-absorbent material 18 (such as polyacrylamide gel particles)saturated with water (or, optionally, with a saturated salt solution) tomaintain the water activity of the air above the chamber 24, and thuswithin the container 20, at a value of 1.00, or the value determined bythe use of the salt solution.

The container 24 may be formed from any suitable material by moulding orby pressing.

The storage arrangement illustrated in FIG. 3 consists of a container30, with an airtight cap or lid 15. A sample 29 of cryptobiotic J3nematodes with their support medium, at the required water activity, iswithin the container 30. The container 30 has a number apertures 31 inits side wall (or in one of its side walls if the horizontalcross-section of the body of the container 30 is rectangular).

A plastic envelope 32, having a front face 33 and a rear face 34,contains (a) polyacrylamide gel particles or starch polyacrylamide gelparticles 18, saturated with water or with a saturated salt solution,and (b) a flexible spacer member 35. The spacer member 35, which mayconveniently be a strip of the material marketed under the trade mark“SCOTCHBRITE”, ensures that the front and rear faces of the envelope donot come into contact with each other during the storage period. Therear face 34 is coated—at least over a region adjacent to the edge ofthe rear face—with an adhesive material which enables the rear face tobe stuck onto the container 30. As shown in FIG. 3, apertures 36 in therear face 34 of the envelope substantially coincide with (that is, atleast partially overlap) the apertures 31 in the container 30. The frontface 33 of the envelope has a plurality of small apertures 37 in it,which permit air to enter the envelope and pass through the apertures 36and 31, and provide a supply of oxygen for the nematodes in the sample29 while maintaining a water activity of 1.00 (or the value establishedby the saturated salt solution) within the container 30.

It will be apparent to persons of skill in this field that theabove-described methods and various forms of apparatus that constitutethe two aspects of the present invention are illustrative only of thoseaspects of the invention, and that variations to and modifications ofthe described and illustrated embodiments may be made without departingfrom the present inventive concepts.

What is claimed is:
 1. A method of preparing third stage infectivejuveniles (J3) of entomopathogenic nematodes of the genera Steinernema(synonym Neoaplectana) and Heterorhabdidtis (synonym Chromonema) forstorage, the method comprising: mixing together an aqueous concentrateof clean J3 entomopathogenic nematodes and substantially anhydrous smallparticles of non-fibrous cellulose, the particles having an averagemaximum dimension which is less than 300 μm, the proportions of theaqueous concentrate and particulate cellulose in the mixture being suchthat (a) sufficient water is absorbed from the concentrate by thecellulose particles to induce cryptobiosis of the nematodes, and (b) themixture, after equilibrating, has a water activity in the range of from0.80 to 0.995.
 2. A method as defined in claim 1, in which the averagemaximum dimension of the cellulose particles is less than 200 μm.
 3. Amethod as defined in claim 1, in which the average maximum dimension ofthe cellulose particles is less than 100 μm.
 4. A method as defined inclaim 1, in which the average maximum dimension of the celluloseparticles is less than 50 μm.
 5. A method as defined in claim 1, inwhich said water activity is in the range of from 0.92 to 0.995.
 6. Amethod as defined in claim 1, in which said water activity is in therange of from 0.95 to 0.99.
 7. A method as defined in claim 1, in whichsaid aqueous concentrate of J3 nematodes includes an anti-fungal agent.8. A method as defined in claim 7, in which said anti-fungal agent isazoxystrobin.
 9. A method of storing third stage infective juveniles(J3) entomopathogenic nematodes prepared for storage by the method ofclaim 1, comprising placing the mixture having the water activity in arange of from 0.80 to 0.995 containing the nematodes in a state ofcryptobiosis and the non-fibrous cellulose particles in a container andmaintaining water activity in the container during storage in the rangeof from 0.80 to 1.0.